Human parainfluenza virus type one (HPIV-1) is a major cause of lower respiratory tract infections (LRI) in infants, young children, and the immunocompromised (Henrickson, K. J., "Lower respiratory viral infections in immunocompetent children," pp. 59-96, In Aronoff SC (ed), Advances in Pediatric Infectious Diseases. Mosby-Year Book, Chicago, Ill., 1994; Henrickson, K. J., et al., Parainfluenza. In: Mandell, Bennet D. (ed) Principles and Practices of Infectious Diseases, Edition 4, Churchill Livingston, N.Y., 1994). This virus has world-wide distribution and probably contributes significantly to childhood mortality in the developing world (Henrickson, K. J., supra, 1994; Henrickson, K. T., et al., supra, 1994). In the United States, we have demonstrated significant morbidity and cost attributable to HPIV-1 epidemics (Henrickson, K. J., et al., "Epidemiology and cost of human parainfluenza virus types one and two infections in young children," Clin. Infect. Dis. 18:770-9, 1994). During these epidemics, approximately 100,000 children less than five years of age are seen in emergency rooms and approximately 35,000 are hospitalized at a combined cost of approximately $90,000,000 (Henrickson, K. J., et al., supra, 1994). Currently, there is no specific therapy or vaccine for any HPIV.
We recently reported that HPIV-1 collected over a 26-year period in a single city demonstrated different genotypes and that one of these genotypes (A) had genotype-specific antigenic markers detectable using MAbs and human sera (Henrickson, K. J., "Monoclonal antibodies to human parainfluenza virus type 1 detect major antigenic changes in clinical isolates," J. Infect. Dis. 164:1128-34, 1991; Henrickson, K. J., et al., "Genetic variation and evolution of human parainfluenza virus type 1 hemagglutinin neuraminidase: Analysis of 12 clinical isolates," J. Infect. Dis. 164:1128-34, 1992). Subsequently, others have found similar antigenic changes in HPIV-1, and one report failed to find genotypes or antigenic markers over a nine-year period (Komada, H., et al., "Antigenic diversity of human parainfluenza virus type 1 isolates and their immunological relationship with Sendai virus revealed by monoclonal antibodies," J. Gen. Virol. 73:875-84, 1992; Hetherington, S. V., et al., "Human parainfluenza virus type 1 evolution combines cocirculation of strains and development of geographically restricted lineages," J. Infect. Dis. 169:248-52, 1994).
HPIV-2 outbreaks occur either biennially or yearly (B. Murphy, et al., "Seasonal pattern in childhood viral lower respiratory tract infections in Melbourne," Med. J. Australia 1:22-24, 1980; M. A. Downham, et al., "Diagnosis and clinical significance of parainfluenza virus infections in children," Arch. Dis. Child 49:8-15, 1974; P. Wright, "Parainfluenza viruses." In: R. B. Belshe ed. Textbook of Human Virology. Littleton, Mass.: PSG Publishing pp. 299-310, 1984), the majority of them appear in fall to early winter. HPIV-2 is a frequent cause of croup. It causes LRI much less frequently than HPIV-1 and HPIV-3, although the difference may be attributable to the difficulties with viral detection. Approximately 60% of HPIV-2 infections take place in the first 5 years of life; the peak incidence occurs in the second year, but significant numbers of infants are infected under 1 year of age. Although frequently overshadowed by HPIV-1 and HPIV-3, HPIV-2 can be predominant in some years (K. J. Henrickson, et al., "Epidemiology and cost of infection with human parainfluenza virus types 1 and 2 in young children," Clin. Infect. Dis. 18:770-779, 1994).
HPIV-3 is unique among the parainfluenaza viruses in its propensity to infect young infants less than 6 months of age. LRI due to HPIV-3 causes approximately 20,000 infants and children to be hospitalized each year in the United States. About 40% of HPIV-3 infections in the first 12 months of life lead to bronchiolitis and pneumonia. It is second only to RSV as a cause of LRI in neonates and young infants. Although endemic throughout the world, this virus also occurs in spring epidemics in North America.
Recent molecular analyses of all four serotypes has revealed more antigenic and genetic heterogeneity than had been appreciated previously (K. J. Henrickson, "Monoclonal antibodies to human parainfluenza virus type 1 detect major antigenic changes in clinical isolates," J. Infect. Dis. 164:1128-1134, 1991; K. J. Henrickson, et al., "Genetic variation and evolution of human parainfluenza virus type 1 hemagglutinin neuraminidase: Analysis of 12 clinical isolates," J. Infect. Dis. 166:995-1005, 1992; K. Prinoski, et al., "Evolution of the fusion protein gene of human parainfluenza virus 3," Virus Res. 22:55-69, 1992; M. Tsurudome, et al., "Extensive antigenic diversity among human parainfluenza type 2 virus isolates and immunological relationships among paramyxoviruses revealed by monoclonal antibodies," Virology 171:38-48, 1989; T. I. Yorlova, et al., "Studies of natural population variability of parainfluenza viruses during their epidemic circulation," Acta Virol. 25:64-70, 1991; K. L. van Wyke Coelingh, et al., "Antigenic variation in the hemagglutinin-neuraminidase protein of human parainfluenza type 3 virus," Virology 143:569-582, 1985; H. Komada, et al., "Strain variation in parainfluenza virus type 4, J. Gen. Virol. 71:1581-1583, 1990; H. Komada, et al., "Antigenic diversity of human parainfluenza virus type 1 isolates and their immunological relationship with Sendai virus revealed by monoclonal antibodies," J. Gen. Virol. 73:875-884, 1992). It appears that all four major HPIV types have virus subgroups that have unique antigenic and genetic characteristics. This includes variability even within HPIV-4 subtypes (H. Komada, et al., supra, 1990). The evolution of these viruses appears to be similar in pattern to influenza B. Most HPIV strains have type-specific antigens that will react in polyclonal serologic testing as previously described. However, HPIV-1 and HPIV-3 have subgroups (A and B) showing progressive antigenic changes (K. J. Henrickson, supra, 1991; K. Prinoski, et al., supra, 1992). Furthermore, HPIV-1 strains isolated over the past 10 years show persistent antigenic and genetic differences compared to the 1957 type strain (K. J. Henrickson, supra, 1991; K. J. Henrickson, supra, 1992; H. Komada, et al., supra, 1992). Because of this, standard reference sera prepared to HPIV isolates from the 1950s, or antigen prepared from these same "type" strains, may not react in current serologic assays.
Detection methods for human parainfluenza viruses 1, 2 and 3 currently include standard viral culture of the suspected infected fluid or tissue. This is a slow and expensive process that may take up to ten days to isolate the virus, and in the best hands, may have a sensitivity of only 40-50%. Direct antigen detection using immunofluorescence is also available both in this country and throughout the world, but the detection rate for HPIV by this method is highly variable with sensitivities averaging only in the 50-70% range and specificities being in the 80-90% range.
A published method concerning the use of an RT-PCR ELISA for the detection of a human parainfluenza virus type-3 was disclosed by Karron in the Journal of Clinical Microbiology (February, 1994, pp. 484-488) entitled "Rapid detection of parainfluenza virus type 3 RNA in respiratory specimens: Use of a reverse transcription-PCR-enzyme-immunoassay." The methods described in this paper are specific for an assay to detect human parainfluenza virus type 3 using specific sequences from the HN gene of HPIV-3. However, their methodology is different from the present invention because the present invention allows for the detection of HPIV-1, 2, and 3 in a single test. Furthermore, the present invention allows for the quantitation of HPIV genomic RNA in a clinical sample.
The published method concerning the use of an RT-PCR-ELISA for the detection of influenza A virus was disclosed by Thomas Cherian in the Journal of Clinical Microbiology (March, 1994, page 623-628) entitled "Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus." The methods described in this paper are specific for an assay to detect influenza A virus using specific sequences from the matrix protein gene. The Cherian method does not allow for the quantitation of influenza A and B virus genomic RNA in a clinical sample.
A published method concerning the use of an RT-PCR ELISA for the detection of respiratory syncytial virus was disclosed by Freymuth in the Journal of Clinical Microbiology (December, 1995, page 3352-3355) entitled "Detection of respiratory syncytial virus by reverse transcription PCR and hybridization with a DNA enzyme immunoassay." The methods described in this paper are specific for an assay to detect RSV using specific sequences from the 1B and N gene. This method does not allow for the detection of RSV, influenza virus, and HPIV in a single test or allow for the quantitation of RSV genomic RNA in a clinical sample.
A fast and efficient method for detection and quantitation in a biological sample of human parainfluenza virus 1, 2 and 3 along with other disease-causing viruses, such as respiratory syncytial virus A and B and influenza virus A and B, is needed.